1. Field of the Invention
The present invention relates to a novel method for measuring a change in a cellular morphological parameter, for example the position and/or the shape of a cell, the method having improved throughput and being compatible with automation.
2. Description of Related Art
Cell motility is an essential element in a variety of normal and aberrant cellular processes. Many cell types exhibit dramatic changes in their morphology, such as movement, elongation of nerve axons and changes in shape during cell division. Wound healing, embryogenesis and immune system responses such as chemotaxis all require specialised cell types to recognise and respond to external stimuli by undergoing cell movement. Investigation of the mechanisms controlling cell movement and development of agents which can modulate cell movement are of significant interest in the development of new therapeutics. Measurement of neurite outgrowth and the effects of candidate drugs on this process are key to evaluating potential treatments for variety of injuries including stroke and spinal cord damage, and for neuro-degenerative conditions such as Parkinson's and Alzheimer's diseases.
A variety of methods have been described for the measurement of cell movement. They fall into two general categories. The first of these relates to methods in which cells are positioned on one side of a membrane barrier and movement of the cells through the barrier is measured (Hagedorn et al, Biochim. Biophys. Acta, (1995), 1269(3), 221-232; Bock and Mrowietz, J. Biochem. Biophys. Methods, (2001), 48(3), 257-268); Dunzendorfer et al, Immunol. Lett., (2000), 71(1), 5-11). The second utilises optical methods for measuring cell movement, which methods typically involve acquiring a series of time lapse images of cells and then performing analysis of the images to measure cell movement (Thurston et al, Cytometry, (1988), 9, 411-417; Thurston et al, Experimental Cell Research, (1986), 165, 380-390).
Major efforts in central nervous system research are focused on the identification of compounds that affect the growth of neurites. Agents that promote nerve growth have therapeutic potential in a wide variety of diseases and traumas, including stroke, spinal cord injuries, and neurodegenerative conditions such as Parkinson's and Alzheimer's diseases. Current methods for measuring neurite outgrowth rely on manual examination of individual cells, or use of complex image analysis methods, to determine the extent of neurite outgrowth from cell bodies (Jiang, Z. G. et al, Brain Res. Dev. Brain Res., (1995), 85(2), 212-9; Patrick, C. W. et al, Exp. Neurol., (1996), 138(2), 277-85). Methods for measuring neurite outgrowth are described in WO 01/11340 by the use of neuronal cells containing a luminescently labeled reporter molecule that reports on cell location, and a second luminescently labeled reporter molecule that reports on neurite outgrowth. Changes in the distribution, environment or activity of the first and second luminescently labelled reporter on or within the cells is determined by digital imaging. This method, while an improvement over image analysis methods based purely on morphology analysis, still requires complex analysis procedures to determine the relative spatial arrangement of the two luminescent reporters.
To date, the methods to measure cellular movement are generally labour intensive to set up, and while variants have been devised using disposable components and chemical analysis methods to determine the number of cells crossing barrier membranes (Frevert et al, J. Immunol. Methods, (1998), 213(1), 41-52), they remain unsuitable to high-throughput applications. Imaging methods have the disadvantage that the analysis process can be extremely complex as sufficient images must be accumulated to enable tracking of individual cells from one image to the next in a series. In addition, since mammalian cells in culture can achieve quite significant relative rates of movement of up to 1-2 μm/minute, (Zicha et al, J. Cell Sci., (1999), 112, 447-454; Thurston and Palcic, Cell Motility and the Cytoskeleton, (1987), 7, 361-367), a single cell of size 10-20 μm can readily move to a position which is non-coincident with its former position in 10-30 minutes. Since cell migration measurements may be performed over many hours or longer, time lapse film or video recording must collect sufficient information to track all cells to avoid misidentification of cells between images. Additionally, once images have been obtained, intensive manual analysis or sophisticated software is required to analyse data and determine the translocation of each cell in a population (Thurston et al, (1988), loc cit; Thurston et al, (1986), loc cit).
Some attempts have been made to simplify assays to make them more suitable for automation in high throughput. These assays have used fluorescent intensity measurements of labelled cells either in standard culture, or in cultures on membrane barriers (Crouch M. F., J. Neurosci. Methods, (2000), 104(1), 87-91), where neurite growth from fluorescently-labelled cells through a membrane is measured. These assays permit quantitation of neurite outgrowth to be performed using fluorescence intensity measurements alone. However, intensity measurements performed on standard cultures cannot accurately discriminate between fluorescently labeled neuronal cell bodies and neurites, leading to inaccuracy in measurement of neurite outgrowth. Barrier methods, while permitting discrimination between cell bodies and neurites, suffer from the same problems in setting up assays as previously described for barrier cell migration methods.